Organoids: development and applications in disease models, drug discovery, precision medicine, and regenerative medicine

Abstract Organoids are miniature, highly accurate representations of organs that capture the structure and unique functions of specific organs. Although the field of organoids has experienced exponential growth, driven by advances in artificial intelligence, gene editing, and bioinstrumentation, a comprehensive and accurate overview of organoid applications remains necessary. This review offers a detailed exploration of the historical origins and characteristics of various organoid types, their applications—including disease modeling, drug toxicity and efficacy assessments, precision medicine, and regenerative medicine—as well as the current challenges and future directions of organoid research. Organoids have proven instrumental in elucidating genetic cell fate in hereditary diseases, infectious diseases, metabolic disorders, and malignancies, as well as in the study of processes such as embryonic development, molecular mechanisms, and host–microbe interactions. Furthermore, the integration of organoid technology with artificial intelligence and microfluidics has significantly advanced large‐scale, rapid, and cost‐effective drug toxicity and efficacy assessments, thereby propelling progress in precision medicine. Finally, with the advent of high‐performance materials, three‐dimensional printing technology, and gene editing, organoids are also gaining prominence in the field of regenerative medicine. Our insights and predictions aim to provide valuable guidance to current researchers and to support the continued advancement of this rapidly developing field.


INTRODUCTION
In 1998 and 2006, the application of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) led to significant advancements in the understanding of mechanisms underlying stem cell fate determination. 1,2In 2009, Clevers et al. 3 first constructed intestinal organoids, sparking a surge of interest in organoid research.Organoid systems can encapsulate numerous essential characteristics of stem cell niches and the tissues they generate, replicating the structure, cellular composition, and self-renewal dynamics of the original tissues. 4][7][8][9] Organoid technology serves as a critical bridge between conventional cell lines and in vivo models.Organoids are widely utilized for studies of disease models, drug discovery, precision medicine, and regenerative medicine. 10,11owever, conventional organoid cultures often lack surrounding stromal cells, immune cells, and vascular endothelial cells.Additionally, variations in induction times and methods for iPSCs, as well as batch inconsistencies in Matrigel, constitute important challenges.Issues related to the maturity and reproducibility of organoids limit their application and development, particularly in disease modeling and regenerative medicine.
In recent years, the integration of artificial intelligence (AI) with high-performance materials and instruments has created new opportunities for organoid applications, offering groundbreaking insights into human physiology and pathology. 12,13When combined with AI technology, microfluidics, and imaging analysis, organoids facilitate rapid functional drug testing and precision medical diagnostics for patients. 14,15Additionally, advancements in hydrogels, biological scaffolds, in vitro vascularization, and tissue engineering have greatly enhanced the role of organoids in regenerative medicine. 16][19] In summary, an understanding of the latest applications and advancements in organoids is crucial, particularly considering their integration with bioengineering, AI technology, and organoid innovation.This review highlights the characteristics of various organoids, engineering methods, and the most recent applications of organoid technology.
We first introduce the characteristics of organoid construction methods and explore how bioengineering approaches can enhance their utility in research and treatment.We then discuss the applications of organoid technology in disease modeling, drug screening, precision therapy, and regenerative medicine.Finally, we highlight the current limitations and future prospects of organoid development, aiming to further enhance their effectiveness in both research and therapy.

DEVELOPMENTS AND ADVANCES IN ORGANOIDS
The self-organizing properties of cells are generally traced back to 1907, when H.V. Wilson demonstrated that sponge cells could self-organize to regenerate an entire organism. 20In 1998 and 2006, the use of hESCs and iPSCs sparked a wave of research into the mechanisms of stem cell fate determination. 1,2In 2009, Clevers et al. 3 demonstrated that the provision of leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) stem cells or isolated crypts with an appropriate niche-consisting of Matrigel, epidermal growth factor, Wingless-related integration site (WNT), Noggin, R-spondin-1, and other cytokines-could lead to the formation of three-dimensional (3D) intestinal organoids.The use of ASC technology to construct organoids laid the foundation for subsequent developments.2][23][24][25] These organoids encapsulate the genotypes, phenotypes, and cellular functions of their respective organs, achieving significant breakthroughs in biological modeling (Figure 1A). 26rom a biomedical perspective, two-dimensional (2D) cell cultures, organoids, multicellular systems, and models ranging from lower animals to higher mammals each summarize bodily functions and processes, spanning from the molecular level to cells, tissues, organs, and whole organisms. 11,27,28Organoids encapsulate the genetic profiles, cellular characteristics, cell-cell interactions, cell-matrix interactions, and physiological functions of organ-specific cells.Their unique features and suitability for scalable culture bridge gaps in existing model systems, thus enhancing our understanding of human development and disease (Figure 1B,C).
Organoids are generated from hESCs, iPSCs, and ASCs, simulating physiological tissue development. 291][32] The cell composition of organoids derived from hESCs and iPSCs is relatively complex, often including mesenchymal, epithelial, and even endothelial components; however, their development is often time consuming.][35][36] ASC-derived organoids are closer in maturity to adult tissues, making them more suitable for the modeling of adult tissue repair and viral infections.In summary, evidence suggests that organoids derived from hESCs, iPSCs, and ASCs can serve as complementary tools in future scientific research and potential personalized medicine.Here, we discuss the development and limitations of organoids according to their germ layer origin (ectoderm: brain, retina; mesoderm: kidney, heart; endoderm: lung, liver, and intestine).Finally, we explore engineering approaches to enhance organoid maturity, reproducibility, and assessment methods.

Advancements and limitations of brain organoids
The human brain comprises a diverse array of cell types and presents complex challenges, including intricate neural circuits, vascular circulation, and immune evasion. 37nimal models and 2D cell cultures often fail to fully address these issues due to ethical concerns and limitations in model complexity. 38However, brain organoids replicate several crucial aspects of early human brain development, encompassing molecular, cellular, structural, and functional dimensions. 26In 2013, Lancaster et al. 24 pioneered the use of iPSC technology to construct brain organoids that exhibited key characteristics of human cortical development, including a distinct progenitor zone rich in radial glial stem cells.In 2018, Mansour et al. 39 successfully transplanted human brain organoids into mouse brains, enabling vascularization and functional integration.In 2020, Pellegrini et al. 40 advanced this work by creating human choroid plexus organoids with selective barriers and cerebrospinal fluid-like secretion in independent compartments.Most recently, in 2023, Schafer et al. 41 constructed vascularized brain organoids containing microglia, effectively modeling the ongoing neuroinflammatory processes observed in the brains of autistic children.
Brain organoids have successfully recapitulated central nervous system viral infections and genetic brain diseases.For example, there is evidence that Zika virus infection causes a reduction in brain organoid size and a loss of surface folds. 42Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in human brain organoids leads to neuron-neuron and neuron-glial cell fusion, resulting in cell death and synaptic loss. 43,44In 2019, Velasco et al. 45 utilized four distinct protocols to produce 3D brain organoids, revealing that early brain region patterning initiates cell specialization and maturation; extrinsic factors also play crucial roles. 45Additionally, serum-treated brain organoids recapitulate Alzheimer's disease-like pathology, inducing levels of β-secretase 1 and glycogen synthase kinase 3 α/β that lead to increased levels of Aβ and p-Tau. 46espite the extensive insights provided by brain organoids, future developments should address several key considerations.First, brain organoids offer a unique opportunity for scientific exploration of consciousness, underscoring the importance of further ethical research to assess potential risks associated with human brain organoids and human-animal chimeric organoids. 47,48econd, although Trujillo et al. 49 found that cortical organoids can generate continuous electrical activity signals, questions remain concerning whether neurons in brain organoids can establish proper synaptic connections to form mature cortical circuits.Third, iPSC-derived brain organoids require further maturation, particularly through the inclusion of essential components (e.g., vascular endothelium, microglia, and the blood-brain barrier) that are critical for the creation of a fully functional brain model. 50

Advancements and limitations of retinal organoids
The retina is a critical structure in the eye responsible for processing photoreceptive information.The differentiation of retinal organoids mirrors the formation of optic vesicles and can include most retinal neuronal cell types, such as cone cells, ganglion cells, bipolar cells, horizontal cells, and amacrine cells. 51In 2011, Eiraku et al. 52 reported the dynamic and autonomous formation of optic cups (retinal primordia) from 3D cultures of mouse embryonic stem cell aggregates.Nakano et al. 53 later discovered that optic cup structures could self-organize in hESC cultures; the neural retina formed a thick layer that spontaneously curved into an apical structure.In 2014, Zhong et al. 54 achieved advanced maturation of photoreceptors in hiPSC-derived retinal organoids, demonstrating the formation of outer segment disks and light sensitivity.In 2019, Achberger et al. 55 combined organoid and organon-chip technologies to create a sophisticated human multilayered tissue retinal platform.This model provided vessel-like perfusion; for the first time, it captured the interactions between in vitro matured photoreceptors and retinal pigment epithelium.
Retinal organoids are a valuable tool for studies of eye development and visual function, as well as the acquisition of retinal tissue.However, they continue to exhibit deficiencies that require improvement.For example, although mature retinal organoids develop photoreceptor outer segment-like structures containing opsins, they do not exhibit proper disk stacking and orientation. 56dditionally, retinal organoids lack vasculature and immune-related cells; they are unable to replicate the foveal structure needed to study diseases such as macular degeneration. 57,58

Advancements and limitations of kidney organoids
Kidney development is believed to originate from the interaction between two embryonic cell populations: ureteric bud and metanephric mesenchyme. 59In 2014, Taguchi et al. 60 and Takasato et al. 60,61 successfully differentiated human iPSCs and hESCs into metanephric mesenchyme organoids and organoids containing nephron structures.Combes et al. 62 utilized single-cell sequencing to compare kidney organoids with human fetal kidneys, revealing a high fidelity in nephron, stromal, and endothelial cell types.Relative to immortalized human podocyte lines, podocytes in kidney organoids demonstrated correct basolateral polarity and gene expression profiles, offering a more accurate representation of in vivo conditions. 63,64onsidering that the kidney is the primary organ responsible for urine concentration, drug metabolism, and toxin filtration, human kidney organoids have been used to simulate autophagy processes in tacrolimus-damaged renal cells, thereby revealing the mechanisms of druginduced nephrotoxicity. 51Concerning pharmacological screening platforms that use organoids, Lawlor et al. 65 in 2021 employed 3D bioprinting technology to precisely manipulate the biophysical properties of kidney organoids and assess the relative toxicities of drugs such as aminoglycosides.
Renal organoid research must address several key points as it progresses toward the creation of a fully functional in vitro kidney model. 66For instance, iPSC-derived kidney organoids face challenges in fully replicating the characteristics of nongenetic kidney diseases due to the removal of epigenetic marks during reprogramming. 67Additionally, although iPSC-derived kidney organoids contain nephron structures, they exhibit limited functionality, growth, and lifespan, likely due to the absence of blood supply, immune cells, and neural cells. 68Furthermore, the complexity of culturing iPSC-derived kidney organoids and tubules poses challenges in maintaining experimental reproducibility.The excessive use of growth factors can also influence immune cell differentiation.For example, A8301 affects T helper cell polarization by altering transforming growth factor-β signaling, 69 and the inclusion of interleukin-2 in T cell cultures impacts podocyte injury and apoptosis. 70

Advancements and limitations of cardiac organoids
The heart is the first functional organ to develop during human embryogenesis, consisting of cardiomyocytes, cardiac fibroblasts, and endothelial cells. 71The unique 3D morphology of cardiac organoids has significantly advanced the field by enabling studies of cavity formation, wall thickness, ejection fraction during contraction, and endothelial tubulogenesis. 72Compared with organoids from other organs, cardiac organoids initially exhibited slower development.In 2017, Giacomelli et al. 73 demonstrated that hiPSCs could codifferentiate into 3D cardiac microtissues composed of cardiomyocytes and endothelial cells.In 2021, Hofbauer et al. 74 utilized hiPSC technology to create the first self-organizing cardiac organoids by regulating the mesodermal WNT-bone morphogenetic protein signaling axis, resulting in chamber-like cardioids with cavities that replicate cardiac lineage structures.In the same year, Lewis-Israeli et al. 75 developed complex luminal structures with multiple cardiac cell types that could reproduce heart field formation and atrioventricular features, reflecting the metabolic disruptions associated with congenital heart defects.Drakhlis et al. 31 reviewed early heart and foregut development, contributing to the investigation of cardiac genetic defects in vitro.Additionally, cardiac organoids have shown promise in transplantation studies.Between 2018 and 2021, several studies demonstrated the transplantation of hESC-and hiPSC-derived cardiac organoids into mouse brain and abdominal cavities. 76Overall, cardiac organoids have rapidly progressed in developmental biology, disease modeling, and transplantation research, gaining substantial attention.
Despite these advancements, cardiac organoids require extensive improvements.For instance, iPSC-derived organoids continue to exhibit embryonic characteristics. 77urrently, they can replicate cell types such as ventricular cardiomyocytes, endothelial cells, and pericytes, but they lack the ability to model the cardiac conduction system that is necessary for studies of arrhythmogenic cardiomyopathy.Additionally, they do not yet incorporate the vascular structures needed to simulate transient ischemic events or the immune cell coculture systems required in analyses of autoimmune interactions. 39,41,78Further research is essential to enhance the complexity of cardiac organoids by applying principles of cardiac development to achieve greater maturation and functional simulation in vitro.

Advancements and limitations of lung organoids
The primary function of the lungs is gas exchange, involving various cell types such as basal cells, club cells, goblet cells, and alveolar epithelial cells (AECs).AEC type I (AEC1) cells are primarily responsible for gas exchange, whereas AEC type II (AEC2) cells secrete surfactants. 79pecific regions from proximal airways to distal alveoli contain distinct stem and progenitor cell populations.In 2013, Barkauskas et al. 80 revealed that AEC2 cells play a key role in alveolar maintenance and repair by forming self-renewing "alveolospheres."Subsequent studies have shown that the SMAD and NOTCH signaling pathways are crucial for airway stem cell proliferation and differentiation, regulating basal cell behavior. 81,82In 2019, Sachs et al. 83 developed human airway organoids that included basal cells, functional multiciliated cells, and mucus-producing secretory cells.Lung organoids have become powerful tools for simulating lung physiology and disease.5][86] Additionally, in 2017, Chen et al. 87 reported that the introduction of Hermansky-Pudlak syndrome 1 mutations into lung organoids could replicate key features of fibrotic lung disease in vitro.The coronavirus disease 2019 (COVID-19) pandemic accelerated the use of lung organoids in infectious disease research.9][90] Organoid models have also been used to study other pathogens, including explorations of the differences during infection and replication of human adenovirus types 3 and 55, assessments of potential antiviral drugs, 91 observations of the replication adaptability of human respiratory syncytial virus and its induced innate cytokine responses in lung organoids, 92 and analyses of avian influenza A virus tropism in human airway organoids, along with changes in cytokine and chemokine profiles. 93espite these advancements, lung organoids have inherent limitations in fully replicating the cell maturity, interactions, and multicellular complexity observed in vivo.In particular, although lung organoids are primarily derived from AEC2 cells, they often lack the conditions necessary for differentiating into the AEC1 cells required for gas exchange. 94

Advancements and limitations of liver organoids
The liver is a crucial hub for numerous physiological processes, including bile metabolism, vitamin metabolism, hormone regulation, blood volume control, immune system support, and endocrine regulation of growth signaling pathways. 95,96The classic method for amplifying liver organoids was established by Huch et al. 97 in 2013; they demonstrated that Lgr5 + cells from injured mouse livers could produce functional liver and bile duct organoids in vitro.In 2015, Huch et al. 98 achieved longterm expansion of bipotent progenitor cells from adult bile ducts in human livers, which could differentiate into functional hepatocytes.They also found that organoids derived from patients with α1-antitrypsin deficiency and Alagille syndrome accurately reflected in vivo pathology and genetic profiles.In 2023, Zhang et al. 99 developed 3D liver bud organoid tissues, reconstructing the structural elements of functional and vascularized organs from liver organoid tissues.Liver organoids have significantly advanced our understanding of various liver diseases; they are valuable for disease mechanism research and drug screening. 100In 2021, Hendriks et al. 101 demonstrated that clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) gene editing could be performed in organoids, facilitating studies of liver development and congenital liver diseases.In the same year, Guan et al. 102 developed a human multilineage liver organoid model exhibiting abnormal bile ducts and fibrosis characteristics.In 2023, Zhang et al. 103 used iPSCs to create three distinct liver organoids for the prediction of drug-induced liver injury based on albumin production, cytochrome P450 expression, and alanine aminotransferase/aspartate aminotransferase release.
Despite rapid advancements in liver organoid research, future improvements may focus on the development of more complex coculture models that include hepatocytes, immune cells, and endothelial cells, as well as more practical and efficient microfluidic devices. 104nother critical area for development is the exploration of transplantation potential in liver organoids.Although Sampaziotis et al. 18 provided evidence in 2021 that bile duct cell organoids could repair bile ducts after transplantation into human livers, additional evidence is needed to ensure the safety of such procedures for actual patient transplants.

Advancements and limitations of intestinal organoids
Intestinal epithelial cells are essential for the absorption and metabolism of drugs, nutrients, and water.Conventional 2D cell lines often fail to accurately replicate the complex characteristics of the gut, such as the distinct genetic profiles of epithelial cells in various regions (e.g., ileum and colon), appropriate pH levels, water absorption, and metabolic products.These limitations hinder their effectiveness in mimicking the true physiological environment within the intestine.In 2009, Clevers et al. 3 successfully created small intestinal organoids in vitro using mouse Lgr5 intestinal stem cells for the first time.In 2021, Qu et al. 105 developed a proliferative intestinal organoid culture system exhibiting complex crypt-villus structures and significantly enhanced regeneration features related to tissue damage.Intestinal organoids retain the histopathological architecture of their tissue of origin.Using organoids, Lindemans et al. 106 found that interleukin-22 promotes epithelial regeneration mediated by intestinal stem cells.Through single-cell RNA sequencing of cancer organoids and colorectal cancer (CRC) patient biopsies, Dmitrieva-Posocco et al. 103 revealed that the ketone body β-hydroxybutyrate acts through the Hcar2-Hopx axis, inhibiting cell viability.Fujii et al. 107 constructed 55 CRC organoids to analyze gene mutation profiles and cellular heterogeneity, showing that the dependency on microenvironmental factors in CRC organoids is highly variable.Organoids not dependent on WNT3A/R-spondin1 often carried mutations in the WNT signaling pathway, including adenomatous polyposis coli, catenin beta 1, and transcription factor 7 like 2.
Intestinal organoids have proven to be valuable tools for studying microbe-epithelium interactions.9][110] These findings have significantly advanced our understanding of the lifecycle of viruses, bacteria, and eukaryotic parasites in the gut, including their processes of adhesion, invasion, infection, and replication.These studies have also provided insights into the cellspecific tropism of these pathogens for intestinal epithelial cells, goblet cells, enteroendocrine cells, and Paneth cells.In 2019, it was found that Cryptosporidium parvum could infect human intestinal and airway organoids, allowing new oocysts to be isolated for downstream analysis. 111dditionally, in 2023, researchers demonstrated that SARS-CoV-2 could infect intestinal organoids; its infection efficiency decreases when the farnesoid X receptor was blocked. 112though intestinal organoid development has made considerable advances, several critical issues must be addressed.First, the 3D spherical structure and extracellular matrix gel used in these models limit drug and microbial access to the lumen, thereby affecting accurate assessment of intestinal permeability and drug absorption. 113Puschhof et al. 114 suggested that bacterial microinjection could be a feasible solution to overcome this barrier.Second, the intestinal epithelium's sensitivity to pH, shear forces from fluid flow, and oxygen gradients poses challenges. 115Although microfluidic technology has been developed to address these factors, the complexity of this technology indicates that its true clinical translation remains a distant goal.Finally, the creation of multicellular organoid systems that replicate the full range of intestinal functions remains a significant challenge.

Challenges and innovations in organoid engineering
The current challenges facing organoids can be summarized as follows.First, most organoids lack vascularization and neural system integration.Second, the determination of cell fate and tissue organization, driven by biochemical factors and the extracellular environment, is impacted by variability in the quality control of Matrigel and cytokines, affecting the standardization and reproducibility of organoids.Finally, the achievement of high-throughput, homogeneous, and standardized production, along with automated operations and intelligent monitoring, assessment, and control of organoids, requires the development of more advanced algorithms and methods.
At present, the engineering of organoids, which leverages bioengineering techniques, biomaterials, and AI technology, is collectively advancing the creation of predictive models, accurate disease simulations, and personalized medical strategies. 13Methods for engineering organoids can be broadly categorized into four areas: engineered cells, engineered microenvironments, engineered detection methods, and engineered cell-cell interactions (Figure 2).
Engineered cells encompass techniques such as CRISPR-Cas9, air-liquid interface, microbial injection, and viral injection.CRISPR/Cas9 has been widely applied in various organoids to study organ embryonic development, 116 tumor suppression, 117 and oncogenic transformation. 118The air-liquid interface method more effectively simulates the physiological environment of organoids.For example, brain organoids cultured at the air-liquid interface show improved neuron survival and The way of engineering organoid.Engineering approaches can be applied to organoids at multiple levels.(A) Engineering the cell includes CRPISPR-Cas9, air-liquid interface, microbial injection, viral infection, and so on.(B) Engineering the microenvironment includes hydrogel, biological scaffolds, adding exogenous substances, and so on.(C) Engineering the detection method includes microfluidics, imaging analysis, and so on.(D) Engineering the cell crosstalk includes 3D bioprinting, multicellular coculture, organoid vascularization, organ-on-a-chip, and so on.axon growth. 119Similarly, air-liquid interface culture allows endometrial organoids to mimic the anatomical structure, cellular composition, hormone-induced menstrual cycle changes, gene expression profiles, and dynamic ciliogenesis of the endometrium. 120Finally, microinjection is used to study epithelial-microbe interactions in organoid research.For instance, the exposure of human intestinal organoids to genotoxic pks + Escherichia coli can induce CRC mutation signatures. 121rganoid cultures primarily rely on extracellular matrices derived from animals or tumors.However, the complex and undefined nature of extracellular matrices sourced from murine tumor stroma limits their use in large-scale drug screening and regenerative medicine applications. 122o address this challenge, Rizwan et al. 123 designed a well-defined viscoelastic hyaluronic acid hydrogel for organoid culture.They discovered that by mimicking the stress relaxation rate of liver tissue, they could induce the growth of cholangiocyte organoids.Additionally, bio-scaffolds offer another approach to the modification of physiological environments within organoids.For example, whereas intestinal organoids cultured in Matrigel typically form randomly developed closed cystic structures, scaffolding can guide the formation of "mini-guts" that more closely resemble physiological states. 124Brain organoids also benefit from improved support structures; Ritzau-Reid et al. 125 developed a microfibril scaffold to guide cavity formation in stem cells and introduced a high-throughput method for the generation, culture, and analysis of engineered brain organoids.Furthermore, the addition of exogenous substances can simulate organoid environments or test developmental processes.For instance, the introduction of oleic acid and free fatty acids to liver organoids can mimic the formation of metabolic-associated fatty liver disease (MAFLD), whereas the addition of troglitazone can simulate druginduced cholestasis. 126The effects of 4-aminopyridine on iPSC-derived brain organoids can also be evaluated in this context. 127espite considerable success in the cultivation of physiologically relevant organoids, the integration of AI, microfluidics, and imaging technologies has accelerated rapid screening, cost-effective extraction of multiscale image features, streamlined multiomics data analysis, and precise preclinical assessment and application.Microfluidic chips have been instrumental in the simulation of high-fidelity disease models and the provision of diagnostic and therapeutic solutions.For example, Abdulla and Quintard 128,129 developed a 3D microfluidic brain organoid platform featuring dynamic fluid perturbations and integrated functional vascularized organoid chips, thereby enhancing organoid growth, maturation, and function.Additionally, AI and imaging technologies have undergone rapid advancement.For instance, Kong et al. 12 utilized machine learning to predict drug responses in colorectal and bladder organoids.Renner et al. 130 combined automated organoid workflows with AI-based analysis, enabling large-scale drug screening for Parkinson's disease and other neurodegenerative diseases.Furthermore, Singaraju et al. 131 developed Organalysis, multifunctional image preprocessing and analysis software for cardiac organoid research, capable of calculating features such as organoid size and fluorescence intensity.
The engineering of cell-cell interactions can effectively address the challenges associated with the construction of organoids of high complexity, size, and structure.Coculture systems provide a relatively straightforward method for the enhancement of organoid complexity and realism.Examples include organoid-fibroblast coculture systems, 132 intestinal organoid-macrophage systems, 133 and liver cancer organoid-endothelial cell systems. 134ioprinting offers a rapid and high-throughput approach to the production of kidney organoids with consistent cell numbers and viability, enabling the assessment of the relative toxicities of drugs such as aminoglycosides. 65A key advantage of 3D bioprinting is its ability to more accurately replicate the tumor microenvironment by allowing precise control over multiple biomaterials, cells, and extracellular matrices within predefined architectures. 135onsidering that drug metabolism and distribution often involve multiple organs, Nguyen et al. 136 developed a multiorgan chip model incorporating human kidney and liver organoids to study the therapeutic effects and distribution of extracellular vesicles.

FOUR APPLICATIONS OF ORGANOIDS
The advent of organoid technology has ushered in a new era in biomedical science.Organoids offer greater clinical fidelity than conventional models and are both cost effective and efficient, enabling more accurate disease simulation and rapid functional testing of drugs.The integration of organoids with synthetic biology, materials science, AI, and gene editing has further accelerated progress in these fields.Currently, organoids are primarily utilized in four key areas: disease modeling, high-throughput drug screening and toxicity assessment, precision medicine, and regenerative medicine (Figure 3).

Organoids for disease models
The development of organoids has provided a clearer perspective on human biology, offering significant advantages Four main applications of organoids.The main applications of organoids include the construction of disease models, drug screening and toxicity assessment, precision medicine, and regenerative medicine.
over conventional disease models (Table 1).Historically, animal models have been the primary tools for studies of disease mechanisms.However, these models face challenges, including genetic and structural differences between species, ethical concerns, long cultivation periods, and high financial costs. 137Although cost effective and readily available, cell lines often display altered genetic backgrounds and limited physiological functionality.Additionally, cell line contamination, as encountered with HeLa cells, 138 LO2 cells, 139 and the MGc80-3 cell line, 140 has been a serious problem for biomedical research.
Patient-derived organoids retain their genetic complexity and functional differences, providing high-fidelity models for studies of organ development, tissue maintenance, and pathogenesis.As shown in Table 2, organoid models have significantly advanced research into various diseases, including genetic disorders, infectious diseases, metabolic conditions, and cancer.

Organoids as advanced models of genetic diseases
Organoids are extensively used to model and study the mechanisms of organ-specific genetic diseases.Organoids derived from ASCs can recapitulate the specific genetic profiles of organs and generally represent relatively mature organ development.For instance, Huch et al. 98 reported in 2015 that liver organoids derived from human ASCs could simulate conditions such as α1-antitrypsin deficiency and Alagille syndrome.Choi et al. 199,200 have utilized inflammatory bowel disease organoids to study the function of the metal ion transporter SLC39A8 and the butyrate-mediated regulation of adherent-invasive E. coli.iPSC-derived organoids tend to be less mature and involve more complex induction processes.However, they are particularly suitable for studies of chromosomal variations, gene mutations, and embryonic development.For example, Li et al. 201 developed a kidney organoid-on-chip model for polycystic kidney disease, discovering that cyst formation is driven by glucose transport into the lumen of the outer epithelial cells.Complex ocular diseases, such as age-related macular degeneration and glaucoma (with >50% heritability), are often poorly replicated by animal models. 202Chirco et al. 203 used retinal organoids with cone-rod homeobox (CRX) mutations to model Leber's congenital amaurosis, whereas Huang et al. 204 showed that retinal organoids could recapitulate key features of X-linked juvenile retinoschisis, including retinal splitting, retinoid production defects, outer segment defects, and impaired ER-Golgi transport.Conventional cell lines and animal models also are not ideal for studies of brain development and genetic brain diseases.Since the establishment of brain organoids in 2013, researchers have used these models to study conditions such as microcephaly, 24 tuberous sclerosis, 205 Down syndrome, 206 and intellectual disabilities and various cortical malformations. 207The advent of brain organoids has enabled researchers to investigate neurological diseases that are challenging to model in animals and to explore disease mechanisms at cellular and molecular levels within a human-specific context.In summary, the emergence of organoid technology has enhanced our understanding of the etiology and pathogenesis of genetic diseases, as well as the drug discovery process.

Organoids as advanced models of infectious diseases
Organoids recapitulate numerous features of in vivo diseases while providing an excellent model for the elucidation of relationships between microorganisms-such as viruses, bacteria, and protozoan parasites-and human respiratory, digestive, and nervous systems.For instance, respiratory and distal alveolar organoids have been used to study infections by Cryptosporidium, 208 influenza virus, 93 and human respiratory syncytial virus, 209 clearly demonstrating the processes of microbial infection and replication in the airways and lungs, while highlighting viral tropism and host epithelial responses.Notably, during the COVID-19 pandemic from 2020 to 2023, organoid-based SARS-CoV-2 research surged, resulting in over 200 publications that continue to have a strong impact.Lamers et al. 210 and Salahudeen et al. 173 found that SARS-CoV-2 can infect human bronchial and alveolar models; alveolar organoids expressing ACE2 receptors facilitated the infection of AEC2 or keratin 5 + basal cell organoids.Additionally, Han et al. 148 identified SARS-CoV-2 inhibitors using lung and colon organoids, including imatinib, mycophenolic acid, and quinacrine dihydrochloride.
As the human organ most exposed to microorganisms, the gastrointestinal tract finds in organoids a valuable platform for exploring mechanisms of microbeepithelium interactions.Helicobacter pylori, a well-known gastric microorganism, was studied in gastric organoids using microinjection techniques by Bartfeld et al. 211 in 2015.In 2022, Cao et al. 212 used patient-derived organoids to discover that H. pylori promotes gastric tumorigenesis by activating the NF-kB-induced RAS protein activator like 2 through the β-catenin signaling axis.Intestinal organoids have also addressed the challenges of replicating and sustaining pathogens such as Shigella flexneri, typhoid, and noroviruses, over-coming limitations of conventional cell and animal models. 110,213he development of brain organoids is crucial for studies of nervous system infections.Brain organoids have been utilized to research diseases caused by human immunodeficiency virus, 214,215 varicella-zoster virus, 216 SARS-CoV-2, 163 Zika virus, 217 and herpes simplex virus, 218 revealing brain-specific viral responses.Additionally, human brain organoids have served as models for large-scale analyses of viral damage.Zhang et al. 219 used organoids to elucidate the effects of influenza viruses (H1N1-WSN and H3N2-HKT68), severe fever with thrombocytopenia syndrome virus, and enteroviruses (EV68 and EV71) on humans.
In summary, organoids are essential for understanding the complex dynamics of pathogen infection, investigating specific mechanisms of pathogen development, and developing effective antipathogen drugs and targets.

Organoids as advanced models of metabolic diseases
Metabolic diseases, such as obesity, diabetes, and cardiovascular diseases, impose a significant burden on modern society.The development of human-specific adipocyte models has been challenging but essential for understanding lipid damage and glucose and lipid metabolism.Taylor et al. 220 developed adipose organoids that incorporate immune cells to study how pro-inflammatory microenvironments stimulate lipolysis in adipocytes under insulin resistance.Escudero et al. 221 created vascularized and functional human beige adipose organoids, which mimic key characteristics of native beige adipose tissue, such as inducible uncoupling protein 1 expression, enhanced uncoupled mitochondrial respiration, and batokine secretion.Furthermore, organoids have been instrumental in the modeling of alcoholic liver disease and MAFLD.Wang et al. 222 developed serum-free liver organoids that simulate alcoholic liver disease pathophysiological changes, including oxidative stress, steatosis, inflammatory mediator release, and fibrosis, upon treatment with ethanol.In 2022, liver organoids from 24 donors were used to demonstrate genetic susceptibility to nonalcoholic steatohepatitis. 2234][225] In 2023, Hendriks et al. 225 utilized human liver organoids in CRISPRbased target discovery and drug screening for steatosis. 225n summary, organoids retain specific pathogenic mutations, providing valuable insights into the underlying mechanisms of metabolic diseases and facilitating the development of personalized medicine.

3.1.4
Organoids as advanced models of cancers Due to the complexity of human tumors, clinical responses to cancer treatments can vary widely.Organoids, particularly those derived from patient-derived ASCs, offer a way to capture this disease heterogeneity.For example, breast cancer organoids often match the histopathology, hormone receptor status, and HER2 status of the original tumors. 143Bladder cancer organoid models retain the genomic alterations present in parental tumors, preserving their heterogeneity. 226Even after long-term culture, gastric cancer organoids maintain a genomic landscape similar to that of in vivo tumors. 227Non-small cell lung cancer organoids preserve the sensitivity of the original tumors to targeted therapies and can form tumors when xenografted into nude mice. 85Organoids have been extensively used to screen anticancer therapies.For instance, Yan et al. 227 demonstrated that gastric cancer organoids are sensitive to several new or investigational drugs, such as abemaciclib, nab-paclitaxel, and the ATR inhibitor VE-822.Pasch et al. 228 showed that organoids can predict sensitivity to chemotherapy and radiation in various cancers, including colorectal, pancreatic, and lung adenocarcinomas.Additionally, organoids are valuable for studies of immunotherapy.They can be cocultured with immune cells, cancer-associated fibroblasts, and other components to recreate the immune microenvironment. 229For example, Kong et al. 230 cocultured tumor-infiltrating lymphocytes with rectal cancer organoids, revealing tumor-infiltrating lymphocyte migration into the organoids and T cell cytotoxicity.Voest et al. 231,232 developed a tumor organoid-T cell coculture system using non-small cell lung cancer and CRC organoids to assess T cell effector functions (including interferon γ secretion and degranulation) and their ability to kill tumor organoids.Cancer-associated fibroblasts interact with cancer cells to enhance tumor heterogeneity, promote tumor growth, and increase resistance to chemotherapy. 233,234rganoids are making substantial progress in cancer research and therapeutic development, addressing critical challenges such as tumor heterogeneity, immune evasion, drug resistance, and metastasis.

Organoids for drug discovery
Organoids have played a pivotal role in the evaluation of new drugs, significantly enhancing the efficiency and accuracy of in vitro validation and testing. 41Compared with primary cells and cell lines, organoids demonstrate superior organ-specific functions and genetic characteristics.Furthermore, they are more cost effective and time efficient than animal models, such as rodents and rhesus monkeys. 235,236atient-derived organoids effectively capture the heterogeneity of patient tumors and facilitate drug screening.For example, Mao et al. 237 constructed CRC organoids to screen 335 drugs, identifying 34 with anti-CRC activity.Fatima and Sun 238,239 also developed colorectal and liver cancer organoids to evaluate the antitumor effects of albendazole and romidepsin, respectively.Shukla et al. 240 assessed the efficacy of 5-fluorouracil and PRIMA-1Met in 3D bioprinted breast cancer models, which improved organoid reproducibility.Organoids are also valuable for the evaluation of drug toxicity in organs such as the liver, kidneys, and heart.Treatments for neurological diseases often rely on neurotherapeutics, but there are significant concerns about their potential adverse effects on reproductive health.Brain chimeroids, brain chimeras, and testicular and ovarian organoids have been used to assess drug toxicity in these contexts. 241,242For instance, Shinozawa et al. 15 reported that iPSC-derived liver organoids exhibited high predictive value (sensitivity: 88.7%, specificity: 88.9%) for drug-induced liver injury.Ding 243 utilized kidney organoids to evaluate the nephrotoxicity of aspirin, penicillin G, and cisplatin.Organoids are also proving useful for toxicity evaluation in nanomedicine research.Li et al. 244 constructed intestinal and hepatic organoid models to assess the cytotoxicity and drugloaded effects of three typical metal-organic frameworks: ZIF-8, ZIF-67, and MIL-125.Additionally, considering the increasing awareness of microplastic hazards, Qin et al. 245 used endometrial organoids to investigate microplastic pollution and its potential impact on reproductive health.
The integration of bioengineering and AI technologies with organoid technology has significantly advanced drug screening and toxicity assessment.Microfluidic technology offers a more physiologically relevant environment for organoids, enabling real-time monitoring of factors such as oxygen tension, cytokine concentrations, and shear stress.For example, Kasendra et al. 246 developed a duodenal small intestine chip that featured polarized cell structures, intestinal barrier function, and expression of key intestinal drug transporters.This model demonstrated improved cytochrome P450 3A4 (CYP3A4) expression and induction compared with Caco-2 cells. 246Zhang et al. 247 designed an automated microfluidic chip-based system for continuous monitoring of organoid drug responses.Whereas single liver organoids can only reflect hepatotoxicity, multiorgan organoid models provide a more comprehensive evaluation of drug-specific organ toxicity and metabolism.In 2017, Skardal et al. 248 constructed a tritissue organ-on-chip system, which included liver, heart, and lung tissues, thereby mimicking the interactive nature of the human body.In 2020, the team further advanced this concept by developing an integrated organoid chip system that incorporated liver, heart, lung, endothelium, brain, and testis organoids to study the metabolic toxicities of drugs such as 5-fluorouracil and ifosfamide. 249I technologies play a crucial role in the analysis of organoid-based data, including microscopic images, transcriptomics, metabolomics, and proteomics, thereby facilitating high-throughput drug screening. 250In 2021, Renner et al. 130 combined automated organoid workflows with AIbased analysis to develop next-generation interdisciplinary high-throughput screening methods for Parkinson's disease and other conditions.In 2022, Matthews et al. 251 introduced a robust image analysis platform capable of automatic identification, labeling, and tracking of individual organoids in brightfield and phase-contrast microscopy experiments, enabling the calculation of dose effects on organoid circularity, solidity, and eccentricity.In 2023, Compte et al. 252 used pancreatic ductal adenocarcinoma organoids combined with AI-driven live-cell image analysis to match retrospective clinical patient responses and screen for chemotherapy drug sensitivity.
The high cost of drug development, estimated at approximately $1 billion per drug, presents significant challenges for pharmaceutical companies.These expenses contribute to elevated drug prices and can inhibit innovation, primarily due to extensive research and development efforts and the high risks associated with clinical trial failures. 253Major companies, including Johnson & Johnson, Roche, Takeda, and Merck, have already initiated investments in high-throughput drug screening using organoids and organ-on-chip technologies. 254,255owever, before organoids can be widely adopted in clinical settings, several critical issues must be addressed by clinicians, pharmacologists, bioengineering laboratories, and regulatory agencies.First, there is a pressing need for more mature and standardized organoid models.Researchers must reach a consensus regarding the specific media, initial cell types, induction methods, and development cycles required for different organoids.Second, the integration of materials science, bioengineering, and AI technologies is crucial to improve the scalability and automation of drug screening processes.This integration will enable more accurate and high-throughput simulations of drug metabolism across various organs, including the skin, intestine, blood vessels, liver, and kidneys.Finally, ethical and regulatory concerns surrounding the use of organoids must be thoroughly addressed.Although organoids pose relatively low ethical risks, they retain patients' genetic information, and the potential for advanced brain organoids to develop consciousness remains a significant concern.

Organoids for personalized medicine
Precision medicine aims to enhance disease characterization at the molecular and genomic levels, thereby improving drug screening.In recent years, extensive organoid biobanks have been established for various organs, including the brain, 164 stomach, 227,256 liver, 257,258 kidneys, 259 intestines 184,260 pancreas, 261 breast, 143 ovaries, 262 cervix, 263 and bladder. 264Organoids derived from tumors and genetic diseases can faithfully replicate the phenotypic and genomic characteristics of primary tumors.For example, Sachs et al. 143 constructed over 100 breast cancer organoids, discovering that the DNA copy number variations and sequence changes in the organoids were consistent with those features in the original tumor tissues.Similarly, Ji et al. 257 developed 65 human liver cancer organoids, with proteomic analysis revealing that the organoids retained the molecular and phenotypic features of the parent cancer tissues.This study demonstrated that the organoid platform could capture both intra-and inter-patient heterogeneity through multiomics analyses, including histopathology, genomics, transcriptomics, and single-cell sequencing.Moreover, organoids are valuable tools for the investigation of rare genetic diseases and identification of personalized treatments.For instance, Yao et al. 265 and Soroka et al. 174 created transcriptional atlases of primary sclerosing cholangitis and cholangiocyte organoids, showing that even in nontumorous conditions, organoids retained the specific pro-inflammatory gene signatures characteristic of primary sclerosing cholangitis.Additionally, Geurts et al. 266 constructed a biobank of intestinal organoids from 664 cystic fibrosis (CF) patients and used CRISPR gene editing to correct CF-related gene mutations in these organoids.
Given the high-fidelity genetic profiles of organoids, drug sensitivity testing with these models can quickly identify the most effective treatments for individual patients.For example, Yuan et al. 267 developed organoids from gallbladder cancer, normal gallbladder tissue, and benign gallbladder adenomas.They found that the dual PI3K/HDAC inhibitor CUDC-907 significantly inhibited the growth of various gallbladder cancer organoids while displaying minimal toxicity to normal gallbladder organoids.Similarly, Phan et al. 268 tested 240 United States Food and Drug Administration (US FDA)-approved or clinically developed protein kinase inhibitors on patient-derived organoids from clear cell/high-grade serous tumors and platinum-resistant high-grade serous ovarian cancer; their findings could aid clinical decision-making.These examples underscore the potential for organoid-based drug sensitivity testing to offer personalized treatment options and advance precision medicine.
This innovative approach aims to streamline the drug discovery process, reduce costs, and increase the success rate of new treatments by providing more accurate models of human physiology and disease.As of July 2024, a search for the keyword "organoids" on ClinicalTrials.govidentified a total of 86 registered clinical trials (Table 3).These trials are predominantly sponsored by institutions or individuals from China and the United States; most have been initiated within the past 5 years.The majority of the trials target cancers; colon cancer (15 trials), breast cancer (16 trials), ovarian cancer (five trials), and gastric cancer (eight trials) are the most common focus areas.These findings underscore the growing momentum in precision medicine research concerning patient-derived organoids.Organoids are emerging as powerful tools for disease gene screening and personalized drug testing, which will facilitate advancements in precision medicine.

Organoids for regenerative medicine
Organ transplantation remains the most definitive and effective treatment for diseases that result in irreversible organ failure.However, due to the shortage of donor organs and the adverse effects of immunosuppressive therapies, thousands of patients die each year while waiting for a transplant. 269The advent of organoid technology has introduced a powerful new tool to the field of regenerative medicine.Organoids have the potential to integrate, mature, vascularize, and develop specific targeted physiological functions in vivo, suggesting that in vitro cultivation of fully functional organs could one day become a reality. 270gnificant progress has been made in the transplantation of human organoids derived from various organs, including the intestine, retina, kidney, liver, pancreas, brain, lung, and heart.Intestinal organoids have been particularly well studied.In 2014, Watson et al. 271 generated human intestinal organoids from hESCs or iPSCs and observed significant expansion and maturation of the epithelium and mesenchyme upon transplantation into mice; the organoids exhibited functional attributes, such as permeability and peptide uptake.Xenotransplanted human ileal organoids retained their regional specificity and formed new villus structures in the mouse colon, offering a potential treatment for patients with short bowel syndrome.In July 2022, a Japanese research team conducted the first autologous transplantation using intestinal organoids cultured from the healthy gut mucosal stem cells of ulcerative colitis patients. 235Retinal organoids have also made substantial progress toward clinical application.Since 2011, iPSC-derived retinal organoids transplanted into animal models of retinal diseases have shown promising results, including the development of mature and functional photoreceptors, integration with the host retina, and improvements in vision and light sensitivity. 272,273In 2023, Hirami et al. 6 transplanted allogeneic iPSC-derived retinal organoid sheets into two patients with advanced retinitis pigmentosa, observing increased retinal thickness at the transplant site without severe adverse events.Organoids derived from cholangiocytes and liver cells have also undergone rapid advancement.Takebe et al. 274 used iPSC technology to create vascularized, functional liver organoids that, when transplanted into immunodeficient mice, quickly formed functional vasculature and exhibited drug metabolism activity.In 2021, Sampaziotis et al. 18 demonstrated that human bile duct organoids maintained their plasticity and restored function in vivo when transplanted back into the human biliary tree, despite the loss of transcriptional diversity in vitro, as shown by singlecell RNA sequencing.In 2022, Willemse et al. 275 enhanced the clinical translation potential of bile duct organoids by using extracellular matrix-derived hydrogels from decellularized human or pig livers for culture, instead of the commonly used tumor-derived basement membrane extract.Other organoids, such as those derived from the brain, pancreas, and lungs, also show regenerative potential.In 2018, Mansour et al. 39 found that brain organoid transplantation led to integration, gradual maturation, neuronal differentiation, synapse formation, and gliogenesis, resulting in the development of a functional vascular system within the grafts.In 2020, Yoshihara et al. 276 reported that human islet-like organoids rapidly reestablished glucose homeostasis in diabetic immunodeficient mice.In 2019, Weiner et al. 277 transplanted dissociated AEC2 organoids into Organoid would enable the prospective identification of "patient tailored optimal treatments." Completed 2017-11-15 NCT04859166

Henan Cancer Hospital
The study first established organoids, followed by drug sensitivity testing to select appropriate clinical treatment options.

Henan Cancer Hospital
The study first established organoids, followed by drug sensitivity testing to select appropriate clinical treatment options.
Recruiting 2024-04- The study aims to validate the therapeutic efficacy of oligonucleotide blockers.
influenza-infected mice, demonstrating that the organoids proliferated and differentiated into AEC2 cells, which were able to support lung regeneration.
Overall, these studies suggest that organoids could become a viable alternative to conventional organ transplantation, potentially alleviating future organ shortages.Table 4 presents a selection of representative papers on human organoid transplantation from the past two decades, highlighting the progress and potential of this emerging field.
Despite their promising potential in regenerative medicine, organoids face several challenges and limitations.First, organoid size and expansion are critical for their clinical application.Transplantable organoids must be as large and numerous as possible, but their proliferation rate is significantly lower than that of cancer cell lines.One feasible approach to address this challenge involves the use of rotating bioreactors.For instance, Qian et al. 217,278 developed a small rotating bioreactor, known as SpinΩ, to optimize brain organoid maturation while reducing costs.Second, organoid maturation presents a significant hurdle.The culture requirements for organoids differ substantially from those for conventional monolayer cell cultures.Most iPSC-and hESC-derived organoids tend to resemble fetal rather than adult cells, which makes it difficult to replicate complete vascularization and neural networks in vitro. 279,280Although scientists have developed various bioreactors to meet the oxygenation, lineage progression, and 3D spatial organization needs of organoids, progress remains challenging. 281Limitations such as high fluid shear and impeller instability can negatively affect shear-sensitive organoid cultures.Although technologies such as SpinΩ have significantly advanced the bioexpression of forebrain organoids, 3D printing technology remains exceedingly complex for widespread use. 282Third, the need for robust preclinical validation models poses another challenge.The immune-deficient models essential for preclinical evaluation are costly, limiting their accessibility and use. 283,284inally, the assurance of transplantation safety is a central requirement.Before organoid transplantation can become a standard practice, concerns regarding potential tumorigenicity and the differentiation capacity of organoids must be addressed. 66The establishment of protocols that comply with Good Manufacturing Practice guidelines to evaluate the long-term risks of in vitrocultured organoids and their potential tumorigenicity post-transplantation offers a rational solution to these safety concerns. 285

Future organoid research
Organoids have made substantial progress in disease simulation, drug development, patient precision medicine, and regenerative medicine.A promising future direction for organoid research lies in the development of multiorganoid systems that use microfluidics and biomaterials to integrate organ systems, thereby enabling highly detailed disease modeling.Perfusable microfluidic devices can ensure the efficient delivery of nutrients, gradient pressure, and oxygen to organoids, which are crucial for the maintenance of their viability and functionality.By manipulating biomaterial parameters such as cell-binding ligands, structural geometry, and mechanical properties, researchers can control organoid development and disease progression. 312,313Because diseases, drugs, and microorganisms impact organ function and structure, as well as the organ's microenvironment, "multiorganoid chips" are poised to become a cutting-edge technology for the evaluation of new drug efficacy and toxicity. 314oreover, the integration of organoid technology, tissue engineering, and AI offers exciting possibilities for enhanced understanding of organoid engineering principles. 13Studies have begun to explore the use of AI in hydrogel design, optimization, and its applications in biomedicine. 315For instance, Kowalczewski et al. 316 utilized unsupervised machine learning to cluster and refine cardiac organoids based on functional similarity, revealing unique features related to their geometric design.Additionally, Lefferts et al. 317 introduced OrgaSegment, a deep learning segmentation model based on MASK-RCNN, which quantifies organoid swelling.This model can differentiate among organoids with various CF transmembrane conductance regulator (CFTR) mutations and assess their responses to CFTR-modulating drugs.The integration of organoid technology with AI has enabled a more comprehensive analysis of organoid behaviors, intricate cellular interactions, and dynamic responses to various stimuli. 318AI is revolutionizing our approach to in vitro modeling, pushing the boundaries of biomedical research.

Challenges and limitations of organoid research
Although the field of organoids holds broad prospects for various applications, it faces several significant challenges and limitations.First, organoid technology bridges the gap between cell lines and in vivo models; however, most organoids lack the surrounding stromal cells, immune cells, and vascular endothelial cells necessary for comprehensive modeling. 319For example, liver organoids often lack hepatocyte zonation and essential components of metabolic fatty liver disease pathogenesis, such as vasculature, immune cells, and neural innervation. 320Second, the organoid industry is hindered by a lack of standardization, a situation exacerbated by the rapid progress in engineered organoids.Factors affecting reproducibility include batchto-batch variation (e.g., patient tissue heterogeneity, as well as timing and method of iPSC induction), culture conditions (e.g., cytokine concentration, matrix gel concentration, and matrix gel composition), and cell composition and organoid structure. 321The resolution of these issues will require collaborative efforts from biomedical scientists, clinicians, and regulatory agencies to standardize and homogenize organoid technology, which would facilitate the transition from scientific research to clinical practice and enable the production of larger-scale organoids for drug screening. 235Third, the cultivation of human organoids often relies on mouse-derived extracellular matrix substitutes, such as Matrigel and basement membrane extracts.However, issues such as the tumor origins of these materials, batch-to-batch variability, high costs, and safety concerns impact the reproducibility and practicality of experimental results.Additionally, the presence of unknown pathogens and potential immune responses make these substrates unsuitable for clinical transplantation. 322][325] Although organoids generally raise fewer ethical issues than other technologies, there are ongoing ethical deliberations concerning potentially controversial entities such as brain organoids and embryoid bodies. 326Much of this debate centers on the ethical status of such organoid models and the implications of their use. 327n summary, although the field of organoids is rapidly advancing, several challenges remain, including issues with standardization, matrix gel contamination, bioethics, and complexity.The resolution of these challenges through the construction of engineered organoids, the development of new materials, and the establishment of industry standards will be crucial for future progress in this field.

Conclusion
In summary, organoids have demonstrated immense potential in disease modeling, drug development, and precision medicine.They are capable of replicating and exhibiting complex biological processes, including multicellular and organ interactions, tumor immune microenvironments, and host-pathogen interactions.Moreover, the integration of organoids with advanced materials science, AI, and gene editing has reshaped our understanding of diseases and translational therapies, significantly advancing our efforts to address and conquer human diseases.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E T H I C S S TAT E M E N T
Not applicable.

F
I G U R E 1 Organoid construction process.(A) Chronological history of organoid establishment for each organ.From left to right in chronological order.(B) Organisms include system hierarchies from the DNA level to the entire body.(C) Model scale from cell lines to large animals such as monkeys.(D) Strategies for forming organoids in vitro.Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), and adult stem cells (AdSC) generate liver, intestine, retinal, blood vessel, brain, skin, cholangiocyte, kidney, and other organoids.
Advantages and disadvantages of organoids compared with other models.
TA B L E 1 Application of organoids in modeling various diseases.Human thyroid follicular organoids have intact hormone production machinery.Both mouse and human thyroid organoids express typical thyroid markers PAX8 and NKX2.1, while the thyroid hormone precursor thyroglobulin is expressed at levels comparable to tissue.
TA B L E 2 tissue.The expression of DTWMK gradually decreased in HT organoids, suggesting that DTYMK may be related to the progression of HT to PTC.In addition, the chemokines CCL2 and CCL3 were significantly highly expressed in HT organoids.168 (Continues) TA B L E 2 (Continued) (Continues) TA B L E 2 (Continued) Patient-derived organoids retain the tissue architecture, gene expression, and genomic landscape of liver tumors and are still able to differentiate into different liver tumor subtypes.
Significant infectious virus particle titers can be detected in human small intestinal organoids readily infected with SARS-CoV-2, and the viral response program is strongly induced.185 (Continues) TA B L E 2 (Continued) (Continues) TA B L E 2 (Continued) 198 Abbreviations: Summary of clinical trials studies for drug testing.
Conceptualization; data curation; formal analysis; writing-original draft: Qigu Yao.Data curation; formal analysis: Sheng Cheng.Data curation; formal analysis: Qiaoling Pan.Investigation: Jiong Yu.Investigation: Guoqiang Cao.Conceptualization: Lanjuan Li.Conceptualization; funding acquisition; writing-review and editing: Hongcui Cao.All authors have read and approved the final version of the manuscript.The figures were created with BioRender.com.This study was supported by Key Research and Development Project of Zhejiang Province (No. 2023C03046), High-Level Personnel Cultivating Project of Zhejiang Province (No. 2023R5243), and Fundamental Research Funds for the Central Universities (No. 2022ZFJH003).
A C K N O W L E D G M E N T S